2 HYBRID QUESTION-Hollenberg system

Dr T.S. Pillay tpillay at nottingham.ac.uk
Tue Nov 9 05:07:00 EST 1999


Dear All,

This question is directed to users of the Hollenberg-BTM116-L40 system:

I am characterising a  two hybrid interaction using the LexA BTM116
plasmid+ GAD424  and the L40 strain and get the following results:
Lets call the proteins X (lexA) and Y (GAL4 activation domain);
The yeast (X+ test proteinY) grow within a few days on His- medium and
almost as well as a good positive control (X + known physiological
interactor).

In the BetaGal filter assay- the positive control (X + known physiological
interactor)  goes blue within an hour but the test protein goes blue
overnight only.

So, my question is:  Is this interaction likely to be physiological if it
mainly turns on the His- reporter. Is it likely to be a low affinity but
possibly still physiologically important? The protein Y itself has
biological relevance to the bait protein X.  I should also point out that
it is specific since when the test protein Y-GAL4AD is tested against
lamin-LexA, nothing happens.

Thanks for any advice.
Dr T.S. Pillay





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